阻断铜离子介导神经毒性的小分子抑制剂研究

过量的铜离子介导淀粉样蛋白异常聚集被认为是导致淀粉样蛋白变性病的主要因素之一. 基于淀粉样蛋白聚集机理及金属铜离子的氧化还原机理, 我们选用β-淀粉样蛋白Aβ1-40作为蛋白模型, 设计并合成出由硫代黄素 T衍生出来的小分子抑制剂. 该小分子抑制剂可以螯合淀粉样蛋白周围过量的一价铜离子, 阻止有害的氧化还原循环过程, 抑制活性氧物质的产生, 从而达到保护细胞的目的. 这为治疗铜离子介导的淀粉样蛋白错误折叠所造成的疾病提供了新的策略.     

Cu(Ⅱ)离子诱导的β-淀粉样蛋白聚集抑制剂筛选及分子构效关系分析

阿尔茨海默症药物的开发对该疾病的治疗非常重要。利用纳米金为探针研究了13种化合物抑制Cu~(2+)诱导的β-淀粉样蛋白聚集的能力,筛选出了10种有效的抑制剂,并获得了抑制剂抑制能力与其分子结构间的关系,利用筛选出的抑制剂实现了β淀粉样蛋白聚集过程的抑制和H_2O_2产生量的减少,对阿尔茨海默症的研究具有重要意义。     

甲状腺结合前清蛋白的理论研究

甲状腺结合前清蛋白TTR是一种具有重要生理功能的蛋白质,它是约30种与淀粉样疾病相关的非同源蛋白中的一种。与TTR相关的淀粉样疾病主要有：家族淀粉化心肌疾病,家族淀粉化神经系统疾病,老年系统性淀粉样病变,以及中枢神经系统选择性淀粉化疾病等。这些疾病是由TTR四聚体解聚过程中错误折叠形成cross-β-sheet结构形态的淀粉样纤维所导致。本文介绍了TTR的生理功能及结构特征,并综述了到目前为止用分子动力学模拟、分子对接和定量构效关系等方法在研究TTR淀粉样机理及TTR和小分子相互作用过程中的计算化学研究成果,为基于TTR结构的TTR淀粉样抑制剂药物分子的设计和筛选提供有力参考。     

氧化异阿朴菲-褪黑素杂合体的合成、抗β淀粉样蛋白聚集及抗氧化活性

基于多靶向策略设计合成了氧化异阿朴菲-褪黑素杂合化合物,测试了它们的抗胆碱酯酶性能及相应的抑制动力学、抗氧化能力和抑制乙酰胆碱酯酶诱导的β-淀粉样蛋白(Aβ)聚集能力。实验结果表明,所合成的化合物对乙酰胆碱酯酶具有中等强度抑制力,其抑制IC50值在微摩尔浓度水平,属于非竞争性抑制剂;对乙酰胆碱酯酶诱导的Aβ淀粉样蛋白聚集的抑制率达到79.3%～84.7%;抗氧化能力是trolox的1.1～1.5倍。     

纳米生物界面对淀粉样蛋白聚集的调控机制研究

本文以阿尔兹海默症的致病多肽β淀粉样蛋白(Aβ)为例,介绍了疾病相关淀粉样蛋白的分子精细结构、组装和聚集过程、聚集体形貌及神经细胞毒性的研究进展,并在此基础上以纳米生物界面对于淀粉样蛋白构象、组装结构、聚集动力学、神经细胞毒性等生物功能的调控机制进行研究.从分子水平上分析和探讨了纳米生物界面与淀粉样蛋白分子或者聚集体的相互作用方式和机理,有助于加深对淀粉样多肽和调节剂之间复杂多样的相互作用方式的理解,对深入了解淀粉样多肽的组装机理和调控机制以及探索治疗神经退行性疾病的药物设计等方面具有较大意义.     

β-淀粉样肽的结构、合成与性质及其与老年痴呆症的关系

β－淀粉样肽是β－淀粉样前体蛋白的剪切产物，分别由39－43个氨基酸残基组成。本文介绍了β－淀粉样肽的化学结构、化学合成及β－淀粉样肽与老年痴呆症的关系。     

硫黄素T对淀粉样β-蛋白质40聚集成核动力学的双重影响

荧光染料硫黄素T常用于淀粉样纤维聚集过程的定性定量检测。虽然有研究表明,某些抑制淀粉样蛋白质聚集的小分子抑制剂会与硫黄素T相互作用,影响其测试结果。但硫黄素T如何影响淀粉样蛋白质的聚集成核动力学尚不清晰。本文以淀粉样β-蛋白质40 (Aβ40)为模型,系统研究了硫黄素T对Aβ40聚集成核的影响。研究发现：硫黄素T能够显著改变Aβ40的聚集成核动力学,且影响程度与硫黄素T的浓度密切相关。即在低浓度硫黄素T存在下,Aβ40成核速率的延迟时间先随着硫黄素T浓度的升高而缩短,后随着硫黄素T浓度的升高延迟时间反而延长。但延伸的速率却随硫黄素T浓度的升高而缓慢增大。另外,硫黄素T基本不会影响Aβ40的二级结构和纤维形态。同时,等温滴定微量热实验结果表明,硫黄素T结合Aβ40之间的主要作用力为疏水相互作用。据此,本研究提出硫黄素T对Aβ40聚集成核动力学的双重影响机理。这些结果有助于进一步了解硫黄素T与淀粉样蛋白质的作用特点,为今后硫黄素T在Aβ40聚集成核动力学实验中的使用提供参考。     

1,4-二巯基苏糖醇对溶菌酶淀粉样纤维化的抑制作用

蛋白质淀粉样纤维化是很多人类疾病的重要特征,筛选蛋白质淀粉样纤维化的抑制剂对于研究和开发相关疾病的治疗药物具有重要意义。本文采用溶菌酶作为模型,探索巯基化合物1,4-二巯基苏糖醇(DTT)对蛋白质淀粉样纤维化的抑制作用。结果表明,DTT对溶菌酶淀粉样纤维化具有较强的抑制作用,其IC50数值为17μmol.L-1。DTT抑制溶菌酶纤维化的作用与其巯基结构有关。在溶菌酶分子高级结构改变产生聚集和纤维化的过程中,DTT分子的巯基通过与溶菌酶的二硫键作用改变了多肽的构象,从而改变了溶菌酶纤维化的进程。     

硫黄素T对淀粉样β-蛋白质40聚集成核动力学的双重影响（英文）

荧光染料硫黄素T常用于淀粉样纤维聚集过程的定性定量检测。虽然有研究表明,某些抑制淀粉样蛋白质聚集的小分子抑制剂会与硫黄素T相互作用,影响其测试结果。但硫黄素T如何影响淀粉样蛋白质的聚集成核动力学尚不清晰。本文以淀粉样β-蛋白质40(Aβ40)为模型,系统研究了硫黄素T对Aβ40聚集成核的影响。研究发现:硫黄素T能够显著改变Aβ40的聚集成核动力学,且影响程度与硫黄素T的浓度密切相关。即在低浓度硫黄素T存在下,Aβ40成核速率的延迟时间先随着硫黄素T浓度的升高而缩短,后随着硫黄素T浓度的升高延迟时间反而延长。但延伸的速率却随硫黄素T浓度的升高而缓慢增大。另外,硫黄素T基本不会影响Aβ40的二级结构和纤维形态。同时,等温滴定微量热实验结果表明,硫黄素T结合Aβ40之间的主要作用力为疏水相互作用。据此,本研究提出硫黄素T对Aβ40聚集成核动力学的双重影响机理。这些结果有助于进一步了解硫黄素T与淀粉样蛋白质的作用特点,为今后硫黄素T在Aβ40聚集成核动力学实验中的使用提供参考。     

功能蛋白纳米材料在环境保护中的应用

蛋白质是一类结构稳定、官能基团丰富的生物大分子。近年来基于功能蛋白纳米材料的改性制备逐渐成为环境领域的研究热点。其中多巴胺、淀粉样纤维和蛋白质杂化纳米花是最具代表性的三类功能蛋白纳米材料。受海洋生物贻贝启发,多巴胺在碱性条件下可氧化自聚成富有黏性的聚多巴胺涂层广泛用于界面改性;淀粉样纤维是功能蛋白经热处理或化学变性形成超高长径比纳米结构,进一步暴露氨基酸活性位点,进而强化对污染物净化性能;而蛋白三维结构也方便与金属磷酸盐形成杂化纳米花结构,提供较大比表面积,可协同金属磷酸盐高效净污。本文基于蛋白质的结构特性,总结了多巴胺、淀粉样纤维和蛋白质杂化纳米花三类纳米复合材料的制备、形成机理及在环境污染控制工程中的应用进展,为后续科研工作提供借鉴。     

Rationally designed amyloid inhibitors based on amyloid-related structural studies

Amyloid proteins correlate with a series of degenerative diseases. Targeting amyloid aggregation has remained a hot topic in therapeutic studies. Numerous inhibitors have been developed, but very few have been approved for marketing. Meanwhile, the growing knowledge of amyloid structural characteristics provides a basis for the rational design of inhibitors. Here we introduce the high-resolution structural findings of amyloid fibrils in recent years and discuss the reported strategies toward rationally designed inhibitors based on amyloid-related structural studies.     

A Hot‐Segment‐Based Approach for the Design of Cross‐Amyloid Interaction Surface Mimics as Inhibitors of Amyloid Self‐Assembly

The design of inhibitors of protein–protein interactions mediating amyloid self‐assembly is a major challenge mainly due to the dynamic nature of the involved structures and interfaces. Interactions of amyloidogenic polypeptides with other proteins are important modulators of self‐assembly. Here we present a hot‐segment‐linking approach to design a series of mimics of the IAPP cross‐amyloid interaction surface with Aβ (ISMs) as nanomolar inhibitors of amyloidogenesis and cytotoxicity of Aβ, IAPP, or both polypeptides. The nature of the linker determines ISM structure and inhibitory function including both potency and target selectivity. Importantly, ISMs effectively suppress both self‐ and cross‐seeded IAPP self‐assembly. Our results provide a novel class of highly potent peptide leads for targeting protein aggregation in Alzheimer’s disease, type 2 diabetes, or both diseases and a chemical approach to inhibit amyloid self‐assembly and pathogenic interactions of other proteins as well.     

Barriers to Small Molecule Drug Discovery for Systemic Amyloidosis

Inhibition of amyloid fibril formation could benefit patients with systemic amyloidosis. In this group of diseases, deposition of amyloid fibrils derived from normally soluble proteins leads to progressive tissue damage and organ failure. Amyloid formation is a complex process, where several individual steps could be targeted. Several small molecules have been proposed as inhibitors of amyloid formation. However, the exact mechanism of action for a molecule is often not known, which impedes medicinal chemistry efforts to develop more potent molecules. Furthermore, commonly used assays are prone to artifacts that must be controlled for. Here, potential mechanisms by which small molecules could inhibit aggregation of immunoglobulin light-chain dimers, the precursor proteins for amyloid light-chain (AL) amyloidosis, are studied in assays that recapitulate different aspects of amyloidogenesis in vitro. One molecule reduced unfolding-coupled proteolysis of light chains, but no molecules inhibited aggregation of light chains or disrupted pre-formed amyloid fibrils. This work demonstrates the challenges associated with drug development for amyloidosis, but also highlights the potential to combine therapies that target different aspects of amyloidosis.     

Monitoring and Targeting the Initial Dimerization Stage of Amyloid Self‐Assembly

Amyloid deposits are pathological hallmark of a large group of human degenerative disorders of unrelated etiologies. While accumulating evidence suggests that early oligomers may account for tissue degeneration, most detection tools do not allow the monitoring of early association events. Here we exploit bimolecular fluorescence complementation analysis to detect and quantify the dimerization of three major amyloidogenic polypeptides; islet amyloid polypeptide, β‐amyloid and α‐synuclein. The constructed systems provided direct visualization of protein‐protein interactions in which only assembled dimers display strong fluorescent signal. Potential inhibitors that interfere with the initial intermolecular interactions of islet amyloid polypeptide were further identified using this system. Moreover, the identified compounds were able to inhibit the aggregation and cytotoxicity of islet amyloid polypeptide, demonstrating the importance of targeting amyloid dimer formation for future drug development.     

Inhibition of amyloid aggregation by formation of helical assemblies

The formation of amyloid aggregates is responsible for a wide range of diseases, including Alzheimer's and Parkinson's disease. Although the amyloid-forming proteins have different structures and sequences, all undergo a conformational change to form amyloid aggregates that have a characteristic cross-β-structure. The mechanistic details of this process are poorly understood, but different strategies for the development of inhibitors of amyloid formation have been proposed. In most cases, chemically diverse compounds bind to an elongated form of the protein in a β-strand conformation and thereby exert their therapeutic effect. However, this approach could favor the formation of prefibrillar oligomeric species, which are thought to be toxic. Herein, we report an alternative approach in which a helical coiled-coil-based inhibitor peptide has been designed to engage a coiled-coil-based amyloid-forming model peptide in a stable coiled-coil arrangement, thereby preventing rearrangement into a β-sheet conformation and the subsequent formation of amyloid-like fibrils. Moreover, we show that the helix-forming peptide is able to disassemble mature amyloid-like fibrils.     

Design of an N-methylated peptide inhibitor of alpha-synuclein aggregation guided by solid-state NMR

Many neurodegenerative diseases are associated with the aggregation of misfolded proteins into amyloid oligomers or fibrils that are deposited as pathological lesions within areas of the brain. An attractive therapeutic strategy for preventing or ameliorating amyloid formation is to identify agents that inhibit the onset or propagation of protein aggregation. Here we demonstrate how solid-state nuclear magnetic resonance (ssNMR) may be used to identify key residues within amyloidogenic protein sequences that may be targeted to inhibit the aggregation of the host protein. For alpha-synuclein, the major protein component of Lewy bodies associated with Parkinson's disease, we have used a combination of ssNMR and biochemical data to identify the key region for self-aggregation of the protein as residues 77-82 (VAQKTV). We used our new structural information to design a peptide derived from residues 77 to 82 of alpha-synuclein with an N-methyl group at the C-terminal residue, which was able to disrupt the aggregation of alpha-synuclein. Thus, we have shown how structural data obtained from ssNMR can guide the design of modified peptides for use as amyloid inhibitors, as a primary step toward developing therapeutic compounds for prevention and/or treatment of amyloid diseases.     

End Capping Alters the Structural Characteristics and Mechanical Properties of Transthyretin (105–115) Amyloid Protofibrils

Pathological amyloid proteins are associated with degenerative and neurodegenerative diseases. These amyloid proteins develop as oligomer, fibrillar, and plaque forms, due to the denatured and unstable status of the amyloid monomers. Specifically, the development of fibrillar amyloid proteins has been investigated through several experimental studies. To understand the generation of amyloid fibrils, environmental factors such as point mutations, pH, and polymorphic characteristics have been considered. Recently, amyloid fibril studies related to end‐capping effects have been conducted to understand amyloid fibril development. However, atomic‐level studies to determine the stability and mechanical properties of amyloid fibrils based on end capping have not been undertaken. In this study, we show that end capping alters the structural characteristics and conformations of transthyretin (TTR) amyloid fibrils by using molecular dynamics (MD) simulations. Variation in the structural conformations and characteristics of the TTR fibrils through end capping are observed, due to the resulting electrostatic energies and hydrophobicity characteristics. Moreover, the end capping changes the mechanical properties of TTR fibrils. Our results shed light on amyloid fibril formation under end‐capping conditions.     

Pro-domain removal in ASP-2 and the cleavage of the amyloid precursor are influenced by pH

Background

One of the signatures of Alzheimer's disease is the accumulation of aggregated amyloid protein, Aβ, in the brain. Aβ arises from cleavage of the Amyloid Precursor protein by β and γ secretases, which present attractive candidates for therapeutic targeting. Two β-secretase candidates, ASP-1 and ASP-2, were identified as aspartic proteases, both of which cleave the amyloid precursor at the β-site. These are produced as immature transmembrane proteins containing a pro-segment.

Results

ASP-2 expressed in HEK293-cells cleaved the Swedish mutant amyloid precursor at different β-sites at different pHs in vitro. Recent reports show that furin cleaves the pro-peptide of ASP-2, whereas ASP-1 undergoes auto-catalysis. We show that purified recombinant ASP-2 cleaves its own pro-peptide at ph 5 but not pH 8.5 as seen by mass spectrometry, electrophoresis and N-terminal sequencing.

Conclusion

We suggest that ASP-2 processing as well as activity are influenced by pH, and hence the cellular localisation of the protein may have profound effects on the production of Aβ. These factors should be taken into consideration in the design of potential inhibitors for these enzymes.     

Identification of amyloidogenic peptide sequences using a coarse-grained physicochemical model

Cross-beta amyloid is implicated in over 20 human diseases. Experiments suggest that specific sequence elements within amyloidogenic proteins play a major role in seeding amyloid formation. Identifying these seeding sequences is important for rationalizing the molecular mechanisms of amyloid formation and for elaborating therapeutic strategies that target amyloid. Theoretical techniques play an important role in facilitating the identification and structural characterization of putative seeding sequences; most amyloid species are not amenable to high resolution experimental structure techniques. In this study we have combined a coarse-grained physicochemical protein model with a highly efficient Monte Carlo sampling technique to identify amyloidogenic sequences in four proteins for which respective experimental peptide fragmentation data exist. Peptide sequences were defined as amyloidogenic if the ensemble structure predicted for three interacting peptides described a stable and regular three-stranded beta-sheet. For such peptides, free energies were calculated to provide a measure of amyloid propensity. The overall agreement between the experimental and predicted data is good, and we correctly identify several self-recognition motifs proposed to define the cross-beta amyloid fibril architectures of two of the proteins. Our results compare very favorably with those obtained using atomistic molecular dynamics methods, though our simulations are 30-40 times faster.     

Multi‐Frequency,Multi‐Technique Pulsed EPR Investigation of the Copper Binding Site of Murine Amyloid β Peptide

Copper‐amyloid peptides are proposed to be the cause of Alzheimer’s disease, presumably by oxidative stress. However, mice do not produce amyloid plaques and thus do not suffer from Alzheimer’s disease. Although much effort has been focused on the structural characterization of the copper‐ human amyloid peptides, little is known regarding the copper‐binding mode in murine amyloid peptides. Thus, we investigated the structure of copper‐murine amyloid peptides through multi‐frequency, multi‐technique pulsed EPR spectroscopy in conjunction with specific isotope labeling. Based on our pulsed EPR results, we found that Ala2, Glu3, His6, and His14 are directly coordinated with the copper ion in murine amyloid β peptides at pH 8.5. This is the first detailed structural characterization of the copper‐binding mode in murine amyloid β peptides. This work may advance the knowledge required for developing inhibitors of Alzheimer’s disease.